Alginate sponge and preparation method thereof

ABSTRACT

The present invention relates to an alginate sponge and a preparation method thereof, more particularly to an alginate sponge having significantly improved flexibility, structural integrity, water-absorptivity, and processability, to be used for medical and tissue engineering purposes, and a simple preparation method thereof. The alginate sponge of the present invention has a maximum bend angle (flexibility) of at least 90°″ , an apparent density (structural integrity) ranging from 0.006 to 0.1 glcm3, and a saline solution absorption ratio ranging from 150 to 700%.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to an alginate sponge and a preparationmethod thereof, and more particularly to an alginate sponge havingsignificantly improved flexibility, structural integrity,water-absorptivity, and processability, to be used for medical andtissue engineering purposes, and a simple preparation method thereof.

(b) Description of the Related Art

Alginic acid is a polysaccharide present in oceanic plants,corresponding to cellulose of land plants. It is a linear-chaincopolymer comprising α-(1→4)-L-guluronic acid and β-(1→4)-D-mannuronicacid.

In general, alginic acid is the main constituent present between cellmembranes of phaeophytae, which are the most productive oceanic plants.Commercially, it is obtained from laminaria, giant kelp, etc. It iswidely used in the form of water-soluble sodium alginate. Water-solublealginate derivatives are widely used as thickening agents, stabilizers,emulsifiers, and microcapsule materials in the fields of food, medicine,and fiber engineering because of their viscosity, biodegradability,non-toxicity, and easy gelation due to multivalent metal ions (e.g.,Ca²⁺).

Additionally, there are many alginate products such as wound dressingsand hemostatics utilizing the biodegradability, moisture absorptivity,hemostaticity, and biocompatibility of alginates.

As for alginate wound dressings, calcium alginate non-woven fiber madeby spinning a sodium alginate solution in a coagulation bath of calciumchloride solution is the most popular. Examples are Kaltostat (Convatec,US), Sorbsan (Bertek, England), Nu-DERM (Johnson & Johnson, US), andTegagen (3M, US). However, the non-woven type alginate wound dressingsleave fiber debris when detached from the wound and are difficult tomake into a variety of shapes. Accordingly, they are not suitable fortissue engineering.

To solve these problems, U.S. Pat. No. 3,653,383, U.S. Pat. No.5,718,916, and U.S. Pat. No. 4,948,575 disclose methods of making analginate sponge by adding a cross-linking agent to an alginate solutionto form an alginate cross-linked gel, and then forming, freezing, andlyophilizing the same. However, the resultant alginate sponge offers badtactility due to its rough surface. Also, it must be thicker than othersponges because its structure is not elaborate, or it has to be replacedfrequently. Moreover, it has poor flexibility and poor adhesivity to awound site, is brittle, has low fluidity, and thus has poorprocessability, so it is difficult to use as a wound dressing.

Accordingly, research on alginate sponges having superior flexibility,structural integrity, water-absorptivity, and processability to be usedin the fields of medicine and tissue engineering are highly required.

SUMMARY OF THE INVENTION

Thus, it is an object of the present invention to provide an alginatesponge having superior flexibility, structural integrity,water-absorptivity, and processability to be used in the fields ofmedicine and tissue engineering.

It is another object of the present invention to provide a method ofpreparing an alginate sponge having significantly improved flexibility,structural integrity, water-absorptivity, and processability to be usedin the fields of medicine and tissue engineering by a simple process.

To attain the objects, the present invention provides an alginate spongehaving a maximum bend angle (flexibility) of at least 90°, an apparentdensity (structural integrity) ranging from 0.006 to 0.1 g/cm³, and asaline solution absorption ratio ranging from 150 to 700%.

The present invention also provides a method of preparing an alginatesponge, comprising the steps of:

a) forming an alginate solution and freezing and lyophilizing it toprepare an alginate sponge medium; and

b) immersing said alginate sponge medium of step a) in a cross-linkingagent solution and washing and drying the same to prepare an alginatesponge.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional optical micrograph of the alginate spongeprepared according to a preferred embodiment of the present invention,which shows the pore distribution.

FIG. 2 is another cross-sectional optical micrograph of the alginatesponge prepared according to a preferred embodiment of the presentinvention, which shows the pore distribution.

FIG. 3 is another cross-sectional optical micrograph of the alginatesponge prepared according to a preferred embodiment of the presentinvention, which shows the pore distribution.

FIG. 4 is a cross-sectional optical micrograph of the alginate spongeprepared according to the conventional method, which shows the poredistribution.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in more detail.

The present inventors worked to develop an alginate sponge havingsuperior flexibility, structural integrity, water-absorptivity andprocessability to be used in the filed of medicine and tissueengineering. In doing so, they found that an alginate sponge prepared byforming an alginate solution and freezing and lyophilizing it to preparean alginate sponge medium, immersing said alginate sponge medium in across-linking agent solution, and then washing and drying it, hassufficient physical properties to be used in the fields of medicine andtissue engineering. That is, it has a maximum bend angle (flexibility)of at least 90°, an apparent density (structural integrity) ranging from0.006 to 0.1 g/cm³, a saline solution absorption ratio ranging from 150to 700%, and significantly improved flexibility, structural integrity,water-absorptivity, and processability.

The present invention is characterized by an alginate sponge having amaximum bend angle (flexibility) of at least 90°, an apparent density(structural integrity) ranging from 0.006 to 0.1 g/cm³, and a salinesolution absorption ratio ranging from 150 to 700%.

If the maximum bend angle is below 90°, the alginate sponge isinsufficiently flexible, so that it cannot be effectively applied on anindented wound site.

Also, if the apparent density is below 0.006 g/cm³, the alginate spongebecomes brittle due to its low structural integrity. Otherwise, if itexceeds 0.1 g/cm³, the surface of the alginate sponge becomes rough andthe flexibility and saline solution absorption ratio decrease.

If the alginate sponge has a maximum bend angle and apparent densitysatisfying the above conditions, its saline solution absorption ratioranges from 150 to 700%. Then, it can be effectively attached andapplied on a wound site, and therefore it becomes a good wound dressing.

The present invention is also characterized by a method of preparing analginate sponge comprising the steps of: forming an alginate solutionand freezing and lyophilizing it to prepare an alginate sponge medium;and immersing said alginate sponge medium in a cross-linking agentsolution and washing and drying it.

The method of preparing an alginate sponge according to the presentinvention is described in more detail.

a) Preparation of Alginate Sponge Medium

In this step, an alginate solution is formed, frozen, and lyophilized toprepare an alginate sponge medium.

Said alginate solution is prepared by dissolving alginate in pure water.

For the alginate, it is preferable to use alginic acid or alkali metalalginate, particularly sodium alginate, potassium alginate, or ammoniumalginate, which are highly soluble in water.

Preferably, said alginate solution has an alginate content ranging from0.5 to 30 wt %. If the alginate content is below 0.5 wt %, the lowproductivity increases production cost. Otherwise, if it exceeds 30 wt%, the high viscosity makes it difficult to remove air bubbles in thesolution and decreases fluidity, so that processability and productionreliability worsen.

Said alginate solution may further comprise a group 1A alkali metalsalt, polyethylene oxide, polyvinyl alcohol, carboxymethylcellulose,carboxylated styrene butadiene latex, polyvinylpyrrolidone, coconut oil,glycerin, or surfactant to improve mechanical properties.

Also, said alginate solution may further comprise such bioactive factorsas fibronectin, vitronectin, acidic fibroblast growth factor FGF, basicFGF, KGF, VEGF, EGF, PDFG-M, PDGF-AB, PDGF-BB, TGF-α, TGF-β, IGF, TNF,GM-CSF, NGF, heparin-binding EGF, interferon, erythropoietin, 1L-1(interleukin-1), IL-2, IL-6, IL-8, and tissue-activated peptide. Saidbioactive factors may be comprised alone or in combination.

Said alginate solution may be formed in a mold or by coating, die-casting, or extrusion. Preferably, said alginate solution is formedafter removing air bubbles. Such formed alginate solution is promptlycooled to −10° C. or lower, and then frozen and lyophilized to preparean alginate sponge medium.

The resultant alginate sponge medium has a fluidity superior to that ofthe conventional cross-linked alginate gel hydrate. Therefore, thealginate sponge prepared therefrom can be processed precisely and poreson the surface and inside of the sponge are fine and uniform, whichimproves flexibility.

b) Preparation of Alginate Sponge

In this step, the alginate sponge medium prepared in step a) is immersedin a cross-linking agent solution for cross-linking, and residualcross-linking agent is washed and dried to prepare an alginate sponge.

For the cross-linking agent comprised in the cross-linking agentsolution, a divalent metal salt or organic cross-linking agent capableof covalent bonding can be used. To be specific, calcium chloride orzinc chloride may be used for the divalent metal salt, andglutaraldehyde, dicyclohexylcarbodiimide, or hexamethylene diisocyanatemay be used for the organic cross-linking agent capable of covalentbonding.

The content of the cross-linking agent comprised in the cross-linkingagent solution is not particularly limited. It can be varied dependingon the requirement and apparent properties of the final alginate spongeproduct.

Also, said cross-linking agent solution may further comprise bioactivefactors mentioned in step a), water-soluble chitosan, hyaluronic acid,pectin, or gelatin to offer anti-bacterial or skin regeneration effects.

After the alginate sponge medium is immersed in the cross-linking agentsolution for cross-linking, it is washed to remove residualcross-linking agent and dried to prepare an alginate sponge havingsuperior processability, flexibility, and uniform and integral pores.

Preferably, the resultant alginate sponge has a maximum bend angle(flexibility) of at least 90°, an apparent density (structuralintegrity) ranging from 0.006 to 0.1 g/cm³, and a saline solutionabsorption ratio ranging from 150 to 700%.

The alginate sponge of the present invention has such superior physicalproperties as flexibility, structural integrity, water-absorptivity, andprocessability in order to be used in the fields of medicine and tissueengineering. According to the present invention, the alginate sponge canbe prepared by a simple process.

Hereinafter, the present invention is described in more detail throughExamples. However, the following Examples are only for the understandingof the present invention and the present invention is not limited by thefollowing Examples.

EXAMPLES Example 1

Sodium alginate having medium viscosity (SIGMA) obtained fromMacrocystis pyrifera (kelp) was dissolved in pure water to 1 wt % toprepare an alginate solution. Air bubbles were completely removed fromthe solution under reduced pressure. Then, the alginate solution was putin a plastic petri dish (diameter=100 mm), frozen at −40° C. andlyophilized to prepare a sodium alginate sponge medium.

The resultant sodium alginate sponge medium was immersed in a 0.2 Mcalcium chloride solution for 30 minutes for cross-linking. Then, it waswashed with deionized water several times to remove residualcross-linking agent. Then, it was frozen at −40° C. again andlyophilized to prepare a water-insoluble cross-linked calcium alginatesponge.

Example 2

A water-insoluble cross-linked calcium alginate sponge was prepared inthe same manner of Example 1, except for using ammonium alginate(CarboMer, US) instead of sodium alginate.

Example 3

A water-insoluble cross-linked calcium alginate sponge was prepared inthe same manner of Example 1, except for using potassium alginate(Kimica, Japan) instead of sodium alginate.

Comparative Example 1

15 mL of 0.2M calcium chloride solution was slowly added dropwise to 300g of the sodium alginate solution (sodium alginate content=1 wt %)prepared in Example 1 while stirring to prepare a cross-linked alginategel. The resultant cross-linked alginate gel was put in a 100 mm plasticpetri dish and frozen at −40° C. and lyophilized to prepare awater-insoluble cross-linked calcium alginate sponge.

Cross-sections of alginate sponges prepared in Examples 1 to 3 andComparative Example 1 were observed with an optical microscope. Theresults are shown in FIGS. 1 to 4. Flexibility, average pore size, andwater absorption ratio were measured as follows. The results are shownin Table 1 below.

a) Flexibility (°)—The maximum bend angle without sponge breaking wasdetermined.

b) Average pore size (g/cm³)—Apparent density was measured and compared.

c) Water absorption ratio (%)—Sponge sample was dried in a desiccator at60° C. for 24 hours and weighed (weight=A). It was immersed in a 0.9%sodium chloride solution at 25° C. for 48 hours. Then, it wascentrifuged at 160 G for 5 minutes and weighed (weight=B). The waterabsorption ratio was determined by the following Equation 1:$\begin{matrix}{{{Water}\quad{absorption}\quad{ratio}\quad(\%)} = {\left\lbrack \frac{\left( {B - A} \right)}{A} \right\rbrack \times 100}} & {{Equation}\quad 1}\end{matrix}$ TABLE 1 Comparative Classification Example 1 Example 2Example 3 Example 1 Flexibility (°) 150 140 145 70 Average pore size0.0377 0.0341 0.0384 0.0046 (g/cm³) Water absorption 306 268 289 X ratio(%)

As seen in Table 1 and FIGS. 1 to 4, the alginate sponges preparedaccording to the present invention (Examples 1 to 3) had superiorflexibility, average pore size (structural integrity), and waterabsorption ratio than the alginate sponge prepared according to theconventional method (Comparative Example 1). In addition, while thealginate sponges of the present invention (Examples 1 to 3) maintain thesponge structure and offer a high saline solution absorption ratio, thealginate sponge of Comparative Example 1 experienced structure breakageas the saline. solution absorption ratio increased.

Therefore, the present invention provides an alginate sponge havingsignificantly improved physical properties such as flexibility,structural integrity, water-absorptivity, and processability to be usedin the fields of medicine and tissue engineering, and which is preparedby a simple process.

While the present invention has been described in detail with referenceto the preferred embodiments, those skilled in the art will appreciatethat various modifications and substitutions can be made thereto withoutdeparting from the spirit and scope of the present invention as setforth in the appended claims.

1. An alginate sponge having a maximum bend angle (flexibility) of at least 90°, an apparent density (structural integrity) ranging from 0.006 to 0.1 g/cm³, and a saline solution absorption ratio ranging from 150 to 700%.
 2. A method of preparing an alginate sponge comprising the steps of: a) forming an alginate solution and freezing and lyophilizing the same to prepare an alginate sponge medium; and b) immersing said alginate sponge medium in a cross-linking agent solution for cross-linking, and washing and drying the same to prepare an alginate sponge.
 3. The method of preparing an alginate sponge of claim 2, wherein said alginate solution of step a) comprises 0.5 to 30 wt % of alginic acid or alkali metal alginate selected from the group consisting of sodium alginate, potassium alginate, and ammonium alginate.
 4. The method of preparing an alginate sponge of claim 2, wherein said alginate solution of step a) further comprises one or more materials selected from the group consisting of alkali metal salt, polyethylene oxide, polyvinyl alcohol, carboxymethylcellulose, carboxylated styrene butadiene latex, polyvinylpyrrolidone, coconut oil, glycerin, and surfactant.
 5. The method of preparing an alginate sponge of claim 2, wherein said alginate solution of step a) further comprises one or more bioactive factors selected from the group consisting of fibronectin, vitronectin, acidic fibroblast growth factor FGF, basic FGF, KGF, VEGF, EGF, PDFG-M, PDGF-AB, PDGF-BB, TGF-α, TGF-β, IGF, TNF, GM-CSF, NGF, heparin-binding EGF, interferon, erythropoietin, 1L-1 (interleukin-1), IL-2, IL-6, IL-8, and tissue-activated peptide.
 6. The method of preparing an alginate sponge of claim 2, wherein said cross-linking agent comprised in said cross-linking agent solution of step b) is a divalent metal salt or an organic cross-linking agent capable of covalent bonding.
 7. The method of preparing an alginate sponge of claim 2, wherein said cross-linking agent solution of step b) further comprises one or more materials selected from the group consisting of a bioactive factor, water-soluble chitosan, hyaluronic acid, pectin and gelatin.
 8. The method of preparing an alginate sponge of claim 2, wherein said alginate sponge has a maximum bend angle (flexibility) of at least 90°, an apparent density (structural integrity) ranging from 0.006 to 0.1 g/cm³, and a saline solution absorption ratio ranging from 150 to 700%. 